Strain ultrasonic elastography imaging features of locally advanced breast cancer: association with response to neoadjuvant chemotherapy and recurrence-free survival.

BACKGROUND: Due to the highly heterogeneity of the breast cancer, it would be desirable to obtain a non-invasive method to early predict the treatment response and survival outcome of the locally advanced breast cancer (LABC) patients undergoing neoadjuvant chemotherapy (NAC). This study aimed at investigating whether strain elastography (SE) can early predict the pathologic complete response (pCR) and recurrence-free survival (RFS) in LABC patients receiving NAC. METHODS: In this single-center retrospective study, 122 consecutive women with LABC who underwent SE examination pre-NAC and after one and two cycles of NAC enrolled in the SHPD001(NCT02199418) and SHPD002 (NCT02221999) trials between January 2014 and August 2017 were included. The SE parameters (Elasticity score, ES; Strain ratio, SR; Hardness percentage, HP, and Area ratio, AR) before and during NAC were assessed. The relative changes in SE parameters after one and two cycles of NAC were describe as ΔA1 and ΔA2, respectively. Logistic regression analysis and Cox proportional hazards model were used to identify independent variables associated with pCR and RFS. RESULTS: Forty-nine (40.2%) of the 122 patients experienced pCR. After 2 cycles of NAC, SR2 (odds ratio [OR], 1.502; P = 0.003) and ΔSR2 (OR, 0.013; P = 0.015) were independently associated with pCR, and the area under the receiver operating characteristic curve for the combination of them to predict pCR was 0.855 (95%CI: 0.779, 0.912). Eighteen (14.8%) recurrences developed at a median follow-up of 60.7 months. A higher clinical T stage (hazard ratio [HR] = 4.165; P = 0.005.), a higher SR (HR = 1.114; P = 0.002.) and AR (HR = 1.064; P <  0.001.) values at pre-NAC SE imaging were independently associated with poorer RFS. CONCLUSION: SE imaging features have the potential to early predict pCR and RFS in LABC patients undergoing NAC, and then may offer valuable predictive information to guide personalized treatment.

Multiparametric Contrast-Enhanced Ultrasound in Early Prediction of Response to Neoadjuvant Chemotherapy and Recurrence-Free Survival in Breast Cancer.

We aimed to explore the value of contrast-enhanced ultrasound (CEUS) in early prediction of pathologic complete response (pCR) and recurrence-free survival (RFS) in locally advanced breast cancer (LABC) patients treated with neoadjuvant chemotherapy (NAC). LABC patients who underwent CEUS before and during NAC from March 2014 to October 2018 were included and assessed. Logistic regression analysis and the Cox proportional hazards model were used to identify independent variables associated with pCR and RFS. Among 122 women, 44 underwent pCR. Molecular subtype, peak intensity (PEAK) and change in diameter were independent predictors of pCR after one cycle of NAC (area under the receiver operating characteristic curve [AUC], 0.81; 95% CI: 0.73, 0.88); Molecular subtype, PEAK and change in time to peak (TTP) were independently associated with pCR after two cycles of NAC (AUC, 0.85; 95% CI: 0.77, 0.91). A higher clinical T (hazard ratio [HR] = 4.75; 95% CI: 1.75, 12.87; p = 0.002) and N stages (HR = 3.39; 95% CI: 1.25, 9.19; p = 0.02) and a longer TTP (HR = 1.06; 95% CI: 1.01, 1.11; p = 0.02) at pre-NAC CEUS were independently associated with poorer RFS. CEUS can be used as a technique to predict pCR and RFS early in LABC patients treated with NAC.

AntiPD-L1 antibody conjugated Au-SPIOs nanoplatform for enhancing radiosensitivity and triggering anti-tumor immune response.

To improve radiotherapy effect by inducing more toxicity for tumors and less for normal tissue and switching immunosuppressive microenvironment caused by expression of PD-L1 and tumor-associated macrophages (TAMs) to immunoreactive microenvironment, we designed a PD-L1-targeted nanoplatform consisting of gold nanoparticles and superparamagnetic iron oxide nanoparticles (antiPD-L1-SPIOs@PLGA@Au). In vivo T2-weighted images, the best contrast effect of tumor was achieved two hours after intravenous injection of antiPD-L1-SPIOs@PLGA@Au. The tumor control caused by irradiation combined with antiPD-L1-SPIOs@PLGA@Au was better than that by radiotherapy alone in clone formation assay and B16F10 subcutaneous tumor model. Radiosensitivity enhancement induced by the addition of antiPD-L1-SPIOs@PLGA@Au was achieved by increasing ROS production and attenuating DNA damage repair. AntiPD-L1-SPIOs@PLGA@Au could promote the polarization of tumor-associated macrophages (TAMs) to M1 and reverse the immunosuppression caused by TAMs. By increasing the expression of CRT in tumor and blocking the PD-L1/PD pathway, antiPD-L1-SPIOs@PLGA@Au with radiation activated the anti-tumor immune response. In conclusion, antiPD-L1-SPIOs@PLGA@Au could be used as a radiosensitizer and a MRI contrast targeting PD-L1, with the functions of blocking the PD-L1/PD-1 immune checkpoint pathway and reversing the immunosuppression caused by TAMs.

Dual-Branch Convolutional Neural Network Based on Ultrasound Imaging in the Early Prediction of Neoadjuvant Chemotherapy Response in Patients With Locally Advanced Breast Cancer.

The early prediction of a patient's response to neoadjuvant chemotherapy (NAC) in breast cancer treatment is crucial for guiding therapy decisions. We aimed to develop a novel approach, named the dual-branch convolutional neural network (DBNN), based on deep learning that uses ultrasound (US) images for the early prediction of NAC response in patients with locally advanced breast cancer (LABC). This retrospective study included 114 women who were monitored with US during pretreatment (NAC pre) and after one cycle of NAC (NAC1). Pathologic complete response (pCR) was defined as no residual invasive carcinoma in the breast. For predicting pCR, the data were randomly split into a training set and test set (4:1). DBNN with US images was proposed to predict pCR early in breast cancer patients who received NAC. The connection between pretreatment data and data obtained after the first cycle of NAC was considered through the feature sharing of different branches. Moreover, the importance of data in various stages was emphasized by changing the weight of the two paths to classify those with pCR. The optimal model architecture of DBNN was determined by two ablation experiments. The diagnostic performance of DBNN for predicting pCR was compared with that of four methods from the latest research. To further validate the potential of DBNN in the early prediction of NAC response, the data from NAC pre and NAC1 were separately assessed. In the prediction of pCR, the highest diagnostic performance was obtained when combining the US image information of NAC pre and NAC1 (area under the receiver operating characteristic curve (AUC): 0.939; 95% confidence interval (CI): 0.907, 0.972; F1-score: 0.850; overall accuracy: 87.5%; sensitivity: 90.67%; and specificity: 85.67%), and the diagnostic performance with the combined data was superior to the performance when only NAC pre (AUC: 0.730; 95% CI: 0.657, 0.802; F1-score: 0.675; sensitivity: 76.00%; and specificity: 68.38%) or NAC1 (AUC: 0.739; 95% CI: 0.664, 0.813; F1-score: 0.611; sensitivity: 53.33%; and specificity: 86.32%) (p<0.01) was used. As a noninvasive prediction tool, DBNN can achieve outstanding results in the early prediction of NAC response in patients with LABC when combining the US data of NAC pre and NAC1.

Her2-Targeted Multifunctional Nano-Theranostic Platform Mediates Tumor Microenvironment Remodeling and Immune Activation for Breast Cancer Treatment.

PURPOSE: The treatment of breast cancer is often ineffective due to the protection of the tumor microenvironment and the low immunogenicity of tumor cells, leading to a poor therapeutic effect. In this study, we designed a nano-theranostic platform for these obstacles: a photothermal effect mediated by a gold shell could remodel the tumor microenvironment by decreasing cancer-associated fibroblasts (CAFs) and promote the release of doxorubicin (DOX) from nanoparticles. In addition, it could realize photoacoustic (PA)/MRI dual-model imaging for diagnose breast cancer and targeted identification of Her2-positive breast cancer. METHODS: Her2-DOX-superparamagnetic iron oxide nanoparticles (SPIOs)@Poly (D, L-lactide-co-glycolide) acid (PLGA)@Au nanoparticles (Her2-DSG NPs) were prepared based on a single emulsion oil-in-water (O/W) solvent evaporation method, gold seed growing method, and carbon diimide method. The size distribution, morphology, PA/MRI imaging, drug loading capacity, and drug release were investigated. Cytotoxicity, antitumor effect, cellular uptake, immunogenic cell death (ICD) effect, and targeted performance on human Her2-positive BT474 cell line were investigated in vitro. BT474/Adr cells were constructed and the antitumor effect of NPs on it was evaluated in vitro. Moreover, chemical-photothermal therapy effect, PA/MRI dual-model imaging, ICD effect induced by NPs, and tumor microenvironment remodeling in human BT474 breast cancer nude mice model were also investigated. RESULTS: Nanoparticles were spherical, uniform in size and covered with a gold shell. NPs had a photothermal effect, and can realize photothermal-controlled drug release in vitro. Chemical-photothermal therapy had a good antitumor effect on BT474/Adr cells and on BT474 cells in vitro. The targeting evaluation in vitro showed that Her2-DSG NPs could actively target and identify Her2-positive tumor cells. The PA/MRI imaging was successfully validated in vitro/vivo. Similarly, NPs could enhance the ICD effect in vitro/vivo, which could activate an immune response. Immunofluorescence results also proved that photothermal effect could decrease CAFs to remodel the tumor microenvironment and enhance the accessibility of NPs to tumor cells. According to the toxicity results, targeted drug delivery combined with photothermal-responsive drug release proved that NPs had good biosafety in vivo. Chemical-photothermal therapy of Her2-targeted NPs has a good antitumor effect in the BT474 nude mice model. CONCLUSION: Our study showed that chemical-photothermal therapy combined with tumor microenvironment remodeling and immune activation based on the Her2-DSG NPs we developed are very promising for Her2-positive breast cancer.

A novel approach with dual-sampling convolutional neural network for ultrasound image classification of breast tumors.

Breast cancer is one of the leading causes of female cancer deaths. Early diagnosis with prophylactic may improve the patients' prognosis. So far ultrasound (US) imaging has been a popular method in breast cancer diagnosis. However, its accuracy is bounded to traditional handcrafted feature methods and expertise. A novel method, named dual-sampling convolutional neural networks (DSCNNs), was proposed in this paper for the differential diagnosis of breast tumors based on US images. Combining traditional convolutional and residual networks, DSCNN prevented gradient disappearance and degradation. The prediction accuracy was increased by the parallel dual-sampling structure, which can effectively extract potential features from US images. Compared with other advanced deep learning methods and traditional handcrafted feature methods, DSCNN reached the best performance with an accuracy of 91.67% and an area under curve of 0.939. The robustness of the proposed method was also verified by using a public dataset. Moreover, DSCNN was compared with evaluation from three radiologists utilizing US-BI-RADS lexicon categories for overall breast tumors assessment. The result demonstrated that the prediction sensitivity, specificity and accuracy of the DSCNN were higher than those of the radiologist with 10 year experience, suggesting that the DSCNN has the potential to help doctors make judgements in clinic.

Targeted gold nanoshelled hybrid nanocapsules encapsulating doxorubicin for bimodal imaging and near-infrared triggered synergistic therapy of Her2-positve breast cancer.

A multifunctional targeted nanoplatform combining photothermal therapy and chemotherapy has emerged as a promising strategy for comprehensive therapies of breast cancer. In this study, we constructed human epidermal growth factor receptor 2 (Her2)-targeted gold nanoshelled poly(lactic-co-glycolic acid) hybrid nanocapsules encapsulating perfluorooctyl bromide, superparamagnetic iron oxide nanoparticles, and doxorubicin (Her2-GPDH nanocapsules) as theranostic agent for bimodal ultrasound/magnetic resonance imaging and synergistic photothermal-chemotherapy of Her2-postive breast cancer cells. Her2-GPDH nanocomposites possessed well-defined spherical morphology, and the average diameter was about 296 nm with good dispersion. Targeting assays demonstrated that Her2-GPDH nanocapsules exhibited higher targeting binding to Her2-positive SKBR3 cells than Her2-negative MDA-MB-231cells. The encapsulation efficiency and the loading content of doxorubicin in Her2-GPDH nanocapsules were 39 ± 1.45% and 3.8 ± 0.52%, respectively, and the agent exhibited pH-responsive and near-infrared light-triggered stepwise release behavior of doxorubicin. In vitro, the agent had potential to serve as feasible candidate for ultrasound imaging and T2-weighted magnetic resonance imaging with a relatively high relaxivity. Cell experiments confirmed that the agent had significant photothermal cytotoxicity on SKBR3 cells, and the combined photothermal-chemotherapy could significantly enhance the anti-tumor effect. In summary, the present Her2-GPDH nanocapsules, a novel multifunctional nanoplatform, will offer a new way for early bimodal molecular-level diagnosis and synergistic treatment of Her2-positve breast cancer.

Her2-Functionalized Gold-Nanoshelled Magnetic Hybrid Nanoparticles: a Theranostic Agent for Dual-Modal Imaging and Photothermal Therapy of Breast Cancer.

Targeted theranostic platform that integrates multi-modal imaging and therapeutic function is emerging as a promising strategy for earlier detection and precise treatment of cancer. Herein, we designed targeted gold-nanoshelled poly (lactic-co-glycolic acid) (PLGA) magnetic hybrid nanoparticles carrying anti-human epidermal growth factor receptor 2 (Her2) antibodies (Her2-GPH NPs) for dual-modal ultrasound (US)/magnetic resonance (MR) imaging and photothermal therapy of breast cancer. The agent was fabricated by coating gold nanoshell around PLGA nanoparticles co-loaded with perfluorooctyl bromide (PFOB) and superparamagnetic iron oxide nanoparticles (SPIOs), followed by conjugating with anti-Her2 antibodies. Cell-targeting studies demonstrated receptor-mediated specific binding of the agent to Her2-positive human breast cancer SKBR3 cells, and its binding rate was significantly higher than that of Her2-negative cells (P < 0.001). In vitro, the agent had capabilities for contrast-enhanced US imaging as well as T2-weighted MR imaging with a relatively high relaxivity (r2 = 441.47 mM-1 s-1). Furthermore, the Her2 functionalization of the agent prominently enhanced the US/MR molecular imaging effect of targeted cells by cell-specific binding. Live/dead cell assay and targeted photothermal cytotoxicity experiments confirmed that Her2-GPH NPs could serve as effective photoabsorbers to specifically induce SKBR3 cell death upon near-infrared laser irradiation. In summary, Her2-GPH NPs were demonstrated to be novel targeted theranostic agents with great potential to facilitate early non-invasive diagnosis and adjuvant therapy of breast cancer.

Quantitative contrast-enhanced ultrasound evaluation of pathological complete response in patients with locally advanced breast cancer receiving neoadjuvant chemotherapy.

PURPOSE: To clarify whether the quantitative parameters of contrast-enhanced ultrasound (CEUS) can be used to predict pathological complete response (pCR) in patients with locally advanced breast cancer receiving neoadjuvant chemotherapy (NAC). MATERIAL AND METHODS: Fifty-one patients with histologically proved locally advanced breast cancer scheduled for NAC were enrolled. The quantitative data for CEUS and the tumor diameter were collected at baseline and before surgery, and compared with the pathological response. Multiple logistic regression analysis was performed to examine quantitative parameters at CEUS and the tumor diameter to predict the pCR, and receiver operating characteristic (ROC) curve analysis was used as a summary statistic. RESULTS: Multiple logistic regression analysis revealed that PEAK (the maximum intensity of the time-intensity curve during bolus transit), PEAK%, TTP% (time to peak), and diameter% were significant independent predictors of pCR, and the area under the ROC curve was 0.932(Az1), and the sensitivity and specificity to predict pCR were 93.7% and 80.0%. The area under the ROC curve for the quantitative parameters was 0.927(Az2), and the sensitivity and specificity to predict pCR were 81.2% and 94.3%. For diameter%, the area under the ROC curve was 0.786 (Az3), and the sensitivity and specificity to predict pCR were 93.8% and 54.3%. The values of Az1 and Az2 were significantly higher than that of Az3 (P = 0.027 and P = 0.034, respectively). However, there was no significant difference between the values of Az1 and Az2 (P = 0.825). CONCLUSION: Quantitative analysis of tumor blood perfusion with CEUS is superior to diameter% to predict pCR, and can be used as a functional technique to evaluate tumor response to NAC.

Ultrasound molecular imaging of breast cancer in MCF-7 orthotopic mice using gold nanoshelled poly(lactic-co-glycolic acid) nanocapsules: a novel dual-targeted ultrasound contrast agent.

BACKGROUND: The development of nanoscale molecularly targeted ultrasound contrast agents (UCAs) with high affinity and specificity is critical for ultrasound molecular imaging in the early detection of breast cancer. PURPOSE: To prospectively evaluate ultrasound molecular imaging with dual-targeted gold nanoshelled poly(lactide-co-glycolic acid) nanocapsules carrying vascular endothelial growth factor receptor type 2 (VEGFR2) and p53 antibodies (DNCs) in MCF-7 orthotopic mice model. METHODS: DNCs were fabricated with an inner PLGA and outer gold nanoshell spherical structure. Its targeting capabilities were evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) in vitro. Contrast-enhanced ultrasound imaging (CEUS) with DNCs was evaluated qualitatively and quantitatively in vitro and in MCF-7 orthotopic mice model by two different systems. The biodistribution of NCs in mice was preliminary investigated. Differences were calculated by using analysis of variance. RESULTS: DNCs showed a well-defined spherical morphology with an average diameter of 276.90±110.50 nm. In vitro, DNCs exhibited high target specificities (79.01±5.63% vs. 2.11±1.07%, P<0.01; 75.54±6.58% vs. 5.21±3.12%, P<0.01) in VEGFR2- and p53-positive cells compared with control cells. In vivo, CEUS displayed a significantly higher video intensity in two systems using DNCs in comparison with non-targeted PLGA@Au NCs and single-targeted NCs. Biodistribution studies revealed that more DNCs in breast cancer tissue could be detected in mice than in other NCs (P<0.05). CONCLUSION: DNCs were demonstrated to be novel dual-targeted UCAs and may have potential applications in early non-invasive visualization of breast cancer.

Preliminary Results of Acoustic Radiation Force Impulse Imaging by Combined Qualitative and Quantitative Analyses for Evaluation of Breast Lesions.

OBJECTIVES: The purpose of this study was to evaluate the application of a new elastographic technique, acoustic radiation force impulse (ARFI) imaging, and its diagnostic performance for characterizing breast lesions. METHODS: One hundred consecutive female patients with 126 breast lesions were enrolled in our study. After routine breast ultrasound examinations, the patients underwent ARFI elasticity imaging. Virtual Touch tissue imaging (VTI) and Virtual Touch tissue quantification (Siemens Medical Solutions, Mountain View, CA) were used to qualitatively and quantitatively analyze the elasticity and hardness of tumors. A receiver operating characteristic curve analysis was performed to evaluate the diagnostic performance of ARFI for discrimination between benign and malignant breast lesions. RESULTS: Pathologic analysis revealed 40 lesions in the malignant group and 86 lesions in the benign group. Different VTI patterns were observed in benign and malignant breast lesions. Eighty lesions (93.0%) of benign group had pattern 1, 2, or 3, whereas all pattern 4b lesions (n = 20 [50.0%]) were malignant. Regarding the quantitative analysis, the mean VTI-to-B-mode area ratio, internal shear wave velocity, and marginal shear wave velocity of benign lesions were statistically significantly lower than those of malignant lesions (all P < .001). The cutoff point for a scoring system constructed to evaluate the diagnostic performance of ARFI was estimated to be between 3 and 4 points for malignancy, with sensitivity of 77.5%, specificity of 96.5%, accuracy of 90.5%, and an area under the curve of 0.933. CONCLUSIONS: The application of ARFI technology has shown promising results by noninvasively providing substantial complementary information and could potentially serve as an effective diagnostic tool for differentiation between benign and malignant breast lesions.

Management of indeterminate pulmonary nodules (<1 cm) newly detected during the follow-up of nasopharyngeal carcinoma patients.

AIM: To investigate the management for the indeterminate pulmonary nodules newly detected during the follow-up for nasopharyngeal carcinoma (NPC) patients. METHODS: Indeterminate pulmonary lesions were identified by searching medical records of NPC patients, who were followed-up with thoracic CT between April 2008 and February 2014. Indeterminate pulmonary nodules were defined as opacities that may be solitary or multiple, which were <1 cm in size and not calcified. Nodules which were followed-up for at least two years, were included into analysis. The nodules with growth were considered as metastatic lesions and those which remained stable or regressed were defined as benign. The relations between the risk of the nodules to develop into metastatic lesions and clinical features and characteristics of nodules were analyzed. RESULTS: Through a median follow-up of 50 months, the majority (81 of 102, 79.4%) had no change in the size of nodules. The nodules have regressed in 11 patients. There were 10 (9.8%) cases with progression at the follow-up. The 10 patients were considered to have lung metastases, for whom continuous increases of lesions were revealed. The volume doubling time of the increasing nodules ranged from 34 to 105 days. The nodules emerging within two years after primary therapies were more likely to develop into metastatic lesions (P = 0.043). CONCLUSION: Ten percent of the indeterminate pulmonary nodules represent metastatic diseases. A short-interval follow-up is recommended and if the nodules remain stable after six months follow-up, it may be proper to prolong the interval of follow-up.

The in vitro study of Her-2 targeted gold nanoshell liquid fluorocarbon poly lactic-co-glycolic acid ultrasound microcapsule for ultrasound imaging and breast tumor photothermal therapy.

Antibody-mediated targeting therapy has been successful in treating patients with breast cancer by improving the specificity and clinical efficacy. In this study, we constructed the human epidermal growth factor receptor-2 (Her2) antibody-conjugated ultrasound contrast agent with lactic-co-glycolic acid (PLGA) as film forming and perfluorocty bromide (PFOB) as internal material, which was coated by gold nanoshell (Her2-PFOB@PLGA@Au), to realize the integration of diagnosis and treatment. The contrast agent was spherical, with the diameter was 256.8 ± 53.4 nm, and had a good dispersion; Ultrasound imaging experiments in vitro showed that the gold nanoshell polylactic acid microcapsule was suitable for ultrasound contrast imaging with the exquisite and uniform dot intensive high echo. The agent had a great photothermal effect under the near-infrared (NIR) with no obvious biological toxicity for both Her2-positive and negative tumor cells; Moreover, both the results of laser scanning confocal microscope (LSCM) and flow cytometer (FCM) demonstrated the great specificity of Her2-PFOB@PLGA@Au conjugating with Her2 positive breast cancer cells (SKBR3). In conclusion, the successful synthesis of the Her2-PFOB@PLGA@Au microcapsule, offered a new therapeutic strategy of combining diagnosis with therapy for fighting against the breast cancer.

Synthesis, characterization, and in vitro evaluation of targeted gold nanoshelled poly(d,l-lactide-co-glycolide) nanoparticles carrying anti p53 antibody as a theranostic agent for ultrasound contrast imaging and photothermal therapy.

Breast cancer is the leading cause of cancer-related deaths in women and earlier detection can substantially reduce deaths from breast cancer. Polymers with targeted ligands are widely used in the field of molecular ultrasound imaging and targeted tumor therapy. In our study, the nanotheranostic agent was fabricated through filling perfluoropropane (C3F8) into poly(d,l-lactic-co-glycolic acid) nanoparticles (PLGA NPs), followed by the formation of gold nanoshell on the surface, then conjugated with anti p53 antibody which has high specificity with the p53 protein overexpressing in breast cancer. The average diameter of the gold nanoshelled PLGA NPs carrying anti p53 antibody (p53-PLGA@Au NPs) was 247 ± 108.2 nm. The p53-PLGA@Au NPs had well-defined spherical morphology and hollow interiors observed by electron microscope, and had a good photothermal effect under the irradiation of an 808 nm laser. The results of laser scanning confocal microscope (LSCM) and flow cytometer (FCM) indicated the specific targeting of p53-PLGA@Au NPs conjugating with breast cancer MCF-7 cells overexpressing p53 protein in vitro. Also the ultrasound imaging experiments in vitro showed that p53-PLGA@Au NPs were suitable for ultrasound contrast imaging. In conclusion, the p53-PLGA@Au NPs are demonstrated to be novel targeted UCAs and may have potential applications in the early diagnosis and targeted near-infrared (NIR) photothermal therapy of breast cancer in the future.

Combined ultrasound-targeted microbubble destruction and polyethylenimine-mediated plasmid DNA delivery to the rat retina: enhanced efficiency and accelerated expression.

BACKGROUND: Gene therapy has potential in the treatment of refractory retinal diseases. It is important to develop an effective delivery system in the retina. The present study aimed to investigate the efficacy and safety of ultrasound (US)-targeted microbubble destruction (UTMD)-mediated polyethylenimine (PEI) to the rat retina. METHODS: Gene transfer was examined by injecting PEI/plasmid DNA (pDNA) with or without microbubbles (MBs) into the subretinal space of rats that were then exposed to US. We investigated enhanced green fluorescent protein (eGFP) expression on flat fundus oculi and performed quantitative analysis. Hematoxylin and eosin staining was used to observe tissue damage. RESULTS: UTMD significantly enhanced PEI/pDNA transfection efficiency safely by increasing both the transgene expression per cell and the percentage of transfected cells of the retina. PEI/pDNA combined with UTMD significantly increased the number of DNA gene copies and the mRNA level in the retinal pigment epithelium (RPE) and neural retina, respectively, compared to PEI/pDNA alone. CONCLUSIONS: The present study demonstrates that enhanced and accelerated pDNA expression can be achieved in the retina/RPE cells in vivo by UTMD physical techniques combined with a PEI chemical vector. Our study provides useful information for further in vivo retinal gene therapy work. Copyright © 2016 John Wiley & Sons, Ltd.

Gene therapy for ocular diseases meditated by ultrasound and microbubbles (Review).

The eye is an ideal target organ for gene therapy as it is easily accessible and immune­privileged. With the increasing insight into the underlying molecular mechanisms of ocular diseases, gene therapy has been proposed as an effective approach. Successful gene therapy depends on efficient gene transfer to targeted cells to prove stable and prolonged gene expression with minimal toxicity. At present, the main hindrance regarding the clinical application of gene therapy is not the lack of an ideal gene, but rather the lack of a safe and efficient method to selectively deliver genes to target cells and tissues. Ultrasound­targeted microbubble destruction (UTMD), with the advantages of high safety, repetitive applicability and tissue targeting, has become a potential strategy for gene­ and drug delivery. When gene­loaded microbubbles are injected, UTMD is able to enhance the transport of the gene to the targeted cells. High­amplitude oscillations of microbubbles act as cavitation nuclei which can effectively focus ultrasound energy, produce oscillations and disruptions that increase the permeability of the cell membrane and create transient pores in the cell membrane. Thereby, the efficiency of gene therapy can be significantly improved. The UTMD­mediated gene delivery system has been widely used in pre­clinical studies to enhance gene expression in a site­specific manner in a variety of organs. With reasonable application, the effects of sonoporation can be spatially and temporally controlled to improve localized tissue deposition of gene complexes for ocular gene therapy applications. In addition, appropriately powered, focused ultrasound combined with microbubbles can induce a reversible disruption of the blood­retinal barrier with no significant side effects. The present review discusses the current status of gene therapy of ocular diseases as well as studies on gene therapy of ocular diseases meditated by UTMD.

Ultrasound-targeted microbubble destruction enhances polyethylenimine-mediated gene transfection in vitro in human retinal pigment epithelial cells and in vivo in rat retina.

The aim of the present study was to investigate the efficacy and safety of ultrasound-targeted microbubble destruction (UTMD)-mediated polyethylenimine (PEI) transfection in cultured human retinal pigment epithelial (RPE) cells in vitro and rat retinas in vivo. An enhanced green fluorescent protein plasmid (pEGFP) was incubated with PEI to prepare a cationic complex (PEI/pEGFP), which was confirmed using a gel retardation assay. In the in vitro study, cultured human RPE cells were subjected to US waves under different conditions with or without microbubbles. The effect of UTMD on the viability of the cells was evaluated. In the in vivo study, gene transfer was examined by injecting PEI/pEGFP into the subretinal space of the rats. The rats treated with PEI/pEGFP and UTMD served as the experimental group, while rats treated with PEI/pEGFP alone served as the control group. The transfected tissue was visualized using an inverted fluorescence microscope. The expression of EGFP was classified into three groups, negative, weak positive and strong positive. Hematoxylin and eosin staining of frozen sections was used to observe tissue damage and the location of the EGFP gene expression. The electrophoresis experiment revealed that PEI treatment was able to condense DNA efficiently. In the in vitro study, the gene transfer efficiency under the optimal UTMD condition was enhanced and significantly higher than control groups. In the in vivo study, UTMD was able to enhance transgene expression in the retina without marked tissue damage. Frozen sections of the optic cups exhibited pEGFP­positive cells, predominantly distributed in the retina. This noninvasive novel combination of UTMD with PEI was able to enhance targeted gene delivery and gene expression in the rat retina without causing any apparent tissue damage, and may be a safe method to transfer genes and drug treatments directly to the retina, therefore being of potential therapeutic value.

Recombinant adeno-associated virus-, polyethylenimine/plasmid- and lipofectamine/carboxyfluorescein-labeled small interfering RNA-based transfection in retinal pigment epithelial cells with ultrasound and/or SonoVue.

The present study was conducted to investigate the efficacy and safety of ultrasound (US)­mediated transfection of the type 2 recombinant adeno­associated virus (AAV) vectors encoding the enhanced green fluorescent protein (EGFP) gene (rAAV), polyethylenimine (PEI)/plasmid EGFP­N1 (pDNA) or lipofectamine (L)/carboxyfluorescein (FAM)­labeled small interfering RNA (siRNA) in the human ARPE­19 retinal pigment epithelial (RPE) cell line, with or without the addition of SonoVue. Cultured RPE cells were exposed to US, with or without SonoVue under different conditions, including variation in the intensity and duration of treatment, and the dose of microbubbles. The effects of ultrasound­targeted microbubble destruction (UTMD) on the structure of pDNA and the transfection ability of rAAV, PEI/pDNA and L/siRNA were also evaluated. Furthermore, the effect of UTMD on RPE cells was evaluated at 0 and 24 h following UTMD. US­mediated transfection (USMT) significantly increased L/siRNA transfection efficiency, as measured by the transgene expression per cell and the percentage of transfected cells. UTMD significantly increased rAAV and PEI/pDNA transfer to RPE cells. UTMD­mediated rAAV or PEI/pDNA delivery was more effective than USMT­mediated delivery of siRNA. Evaluating cell viability at 24 h post­UTMD provided more valuable information than immediate evaluation following UTMD. Furthermore, there was minimal cytotoxicity and minimal change to the structure of pDNA under the optimal parameters. UTMD/US may be of use in enhancing rAAV, PEI/pDNA and L/siRNA transgene expression of ARPE­19 cells in vitro. Studies on the transfection of different nucleotides (such as pDNA and siRNA) and different types of vectors (chemical and biological) mediated by UTMD may provide useful information to guide future in vivo and transfection studies.

Enhanced downregulation of transforming growth factor­ß2 in rat retinal pigment epithelium cells by adeno­associated virus­mediated ribonucleic acid interference combined with ultrasound or microbubbles.

The present study was designed to determine the efficiency and safety of ultrasound (US) and/or US contrast agent microbubbles (MBs) in the delivery of type 2 recombinant adeno-associated virus­delivered transforming growth factor­ß2 short hairpin ribonucleic acid encoding the enhanced green fluorescent protein gene (rAAV2­TGFß2 shRNA­EGFP) and the downregulation of TGFß2 in rat retinal pigment epithelium (RPE­J) cells. The effects of US and/or MBs on the delivery of rAAV2­EGFP and rAAV2­TGFß2 shRNA­EGFP were evaluated by fluorescence microscopy and flow cytometry. The potential toxicity of cell viability under various US or MB conditions was assessed by CellTiter 96® AQueous One solution cell proliferation assay. The level of TGFß2 mRNA in RPE­J cells under various conditions was estimated by reverse transcription­quantitative polymerase chain reaction analysis. The results obtained demonstrated that low-intensity US (0.5 W/cm2 and 30 sec) or SonoVue (MB:cell ratio, 40:1) increased the delivery efficiency of rAAV2­EGFP and rAAV2­TGFß2 shRNA­EGFP to RPE­J cells, whereas the combination of US with MBs did not further increase but instead decreased rAAV transfection. Under the optimal conditions of rAAV delivery, enhanced TGFß2 gene silencing with a combination of US or SonoVue with rAAV2­TGFß2 shRNA resulted in a significant decrease in mRNA levels compared with rAAV2­TGFß2 shRNA alone. US or SonoVue was used safely to enhance the delivery of rAAV2­TGFß2 shRNA to RPE­J cells. A combination of the biological (rAAV2­TGFß2 shRNA) and physical (US or SonoVue) approaches downregulated the mRNA level of TGFß2 more effectively.

Diagnostic efficacy of contrast-enhanced sonography by combined qualitative and quantitative analysis in breast lesions: a comparative study with magnetic resonance imaging.

OBJECTIVES: The purpose of this study was to evaluate the diagnostic efficacy of contrast-enhanced sonography for differentiation of breast lesions by combined qualitative and quantitative analyses in comparison to magnetic resonance imaging (MRI). METHODS: Fifty-six patients with American College of Radiology Breast Imaging Reporting and Data System category 3 to 5 breast lesions on conventional sonography were evaluated by contrast-enhanced sonography and MRI. A comparative analysis of diagnostic results between contrast-enhanced sonography and MRI was conducted in light of the pathologic findings. RESULTS: Pathologic analysis showed 26 benign and 30 malignant lesions. The predominant enhancement patterns of the benign lesions on contrast-enhanced sonography were homogeneous, centrifugal, and isoenhancement or hypoenhancement, whereas the patterns of the malignant lesions were mainly heterogeneous, centripetal, and hyperenhancement. The detection rates for perfusion defects and peripheral radial vessels in the malignant group were much higher than those in the benign group (P < .05). As to quantitative analysis, statistically significant differences were found in peak and time-to-peak values between the groups (P < .05). With pathologic findings as the reference standard, the sensitivity, specificity, and accuracy of contrast-enhanced sonography and MRI were 90.0%, 92.3%, 91.1% and 96.7%, 88.5%, and 92.9%, respectively. The two methods had a concordant rate of 87.5% (49 of 56), and the concordance test gave a value of κ = 0.75, indicating that there was high concordance in breast lesion assessment between the two diagnostic modalities. CONCLUSIONS: Contrast-enhanced sonography provided typical enhancement patterns and valuable quantitative parameters, which showed good agreement with MRI in diagnostic efficacy and may potentially improve characterization of breast lesions.